Aao6135 Gibcus SM

www.sciencemag.org/cgi/content/full/science.
aao6135/DC1
Supplementary Materials for

A pathway for mitotic chromosome formation
Johan H. Gibcus,* Kumiko Samejima,* Anton Goloborodko,* Itaru Samejima, Natalia
Naumova, Johannes Nuebler, Masato T. Kanemaki, Linfeng Xie, James R. Paulson,
William C. Earnshaw,† Leonid A. Mirny,† Job Dekker†
*These authors contributed equally to this work.
†Corresponding author. Email: bill.earnshaw@ed.ac.uk (W.C.E.); leonid@MIT.edu (L.A.M.);
job.dekker@umassmed.edu (J.D.)
Published 18 January 2018 on Science First Release

DOI: 10.1126/science.aao6135
This PDF file includes:
Materials and Methods

Figs. S1 to S27
Captions for Tables S1 to S4
Caption for Movie S1
References
Other Supplementary Material for this manuscript includes the following:

(available at www.sciencemag.org/cgi/content/full/science.aao6135/DC1)
Tables S1 to S4
Movie S1
Materials and Methods
Cell culture
Chicken DT40 (B-cell lymphoma) cells were cultured in RPMI 1640 medium supplemented
with 10 % fetal bovine serum and 1% chicken serum at 39°C in 5% CO2 in air.
Stable transfection into DT40 cells was performed as described previously (23). Transient
transfection into DT40 cells was performed using a Neon transfection system from
ThermoFisher Scientific. 2-4 million cells suspended in 100 µl 1X buffer R were mixed with
plasmid DNA (4-10 µg), then electroporated at setting 5.
Doxycycline (BD) dissolved in water (1mg/ml) was added to a final concentration of 0.5
µg/ml. For G2 arrest, 1NM-PP1 dissolved in DMSO (10 mM) was added to cultures at a final
concentration of 2 µM. Degradation of AID-containing proteins was induced by addition of a 50
mM solution of Indole-3-acetic acid (auxin, Fluka) dissolved in ethanol to a final concentration
of 125 µM. To prevent cells from entering anaphase, Nocodazole (Sigma-Aldrich) dissolved in
DMSO at 1 mg/ml was added to some cultures to a final concentration of 0.5 µg/ml (see details
below, Table S1).
Cell lines
A summary of the cell lines described below can be found in Table S3.
SMC2-AID-GFP cells: SMC2-AID-GFP cells were derived from a previously described
SMC2 conditional (doxycycline induced) knockout cell line (54). Plasmids coding SMC2-
mAID-GFP and OsTIR1 were randomly integrated into the DT40 genome. A 3.8 kb fragment of
the endogenous GgSMC2 promoter (54) drove expression of the GgSMC2 cDNA fused with a
minimal AID (mAID) tag (AtIAA1765-132) and GFP tag (SMC2-mAID-GFP) at the C-
terminus. A CMV promoter drove the expression of plant-specific F-box protein OsTIR1 linked
to a MmDHFR cDNA by a T2A peptide (synthesized at Thermofisher Scientific and cloned into
pCDNA3). 10 µM Methotrexate (MTX) was used to select cells expressing high levels of
MmDHFR and OsTIR1. The resultant cell lines (named SMC2-AID-GFP) were cultured
continuously in the presence of doxycycline (0.5 µg/ml) to suppress the expression of non-
tagged GgSMC2 protein.
CAP-H-AID-GFP cells: Plasmids encoding CAP-H-mAID-GFP and plant specific F-box
protein OsTIR1 were randomly integrated into the genome of a CAP-H conditional (doxycycline
induced) knockout cell line (61). A CMV promoter drove the expression of the GgCAP-H-
mAID-GFP. A CMV promoter drove the expression of OsTIR1 linked to a MmDHFR cDNA by
a T2A peptide (synthesized at Thermofisher Scientific and cloned into pCDNA3). 10 µM
Methotrexate (MTX) was used to select cells expressing high levels of MmDHFR and OsTIR1.
The resultant CAP-H-AID-GFP cell lines were cultured continuously in the presence of
doxycycline (0.5 µg/ml) to suppress the expression of non-tagged GgCAP-H protein.
CAP-H2-AID-GFP cells: CAP-H2-AID-GFP cells were constructed starting with wild type
DT40 cells. A codon-optimized GgCAP-H2 cDNA was synthesized (Thermofisher Scientific)
and cloned into pAID2.3C so that CAP-H2 was tagged with mAID and GFP at its C-terminus. A
CMV promoter drove the co-expression of OsTIR1 and CAP-H2-mAID-GFP, which are linked
by a P2A peptide in the pAID2.3C-CAP-H2 plasmid. The pAID2.3C-CAP-H2 plasmid was
randomly integrated into the DT40 genome. The endogenous GgCAP-H2 gene was then
inactivated by transient transfection of plasmids encoding hCas9 cDNA and GgCapH2 guide
RNA (Thermofisher Scientific). The target sequence of guide RNA was
CCTATACTCGCTGGTCTACCAGG.
CDK1as cells: A CMV promoter drove the expression of a XlCdk1as cDNA (22) linked to
a puromycin or zeocin resistance gene via a T2A peptide. This construct was integrated at
random in the genome of the desired target cell line. The endogenous GgCdk1 gene was then
inactivated by transient transfection of plasmids encoding hCas9 cDNA (Addgene #41815) and
GgCdk1 guideRNA (based on Addgene #41824). The target sequence of the guide RNA was
AAAATACGTCTAGAAAGTG. SMC2-AID-GFP cells, CAP-H-AID-GFP cells and CAP-H2-
AID-GFP cells were individually converted into CDK1as cell lines by this method. Wild type
CDK1as cells were a gift of Helfrid Hochegger (University of Sussex) (22).
Detailed protocols to establish DT40-AID/CDK1as cell lines are available upon request to
Dr. Kumiko Samejima.
Time course experiments

Typically 1-3 x107 CDK1as wild type cells/sample were treated with 1NM-PP1 (final
concentration: 2 µM) for 10 h, after which over 95% were in G2 (Table S1). G2-arrested cells
were collected prior to 1NM-PP1-washout. To release cells from the G2 arrest, 1NM-PP1 was
washed out by collecting the cells by centrifugation (1300 rpm, 2-5 min - depending on the
volume of cultured cells - at RT) and suspending the cells in fresh media (10-20 ml). Cells were
washed twice over a period of 10-20 minutes (this varied depending on the number of cultures
being processed simultaneously). These cells were suspended in fresh media and divided into
flasks (one flask for each time point, typically 1-3 x107 cells per flask) and returned to the
incubator (counted as t = 0 minutes). At each time point (0, 2.5, 5, 7.5, 10, 30, 45, 60, 75
minutes), a flask was removed from the incubator and cells with media were transferred to a 50
ml Falcon tube, which was topped up with fresh media to 45 ml.
Three ml of 16% formaldehyde solution was added to the 50 ml tube to obtain a final
concentration of 1%. The content was quickly mixed by inverting the tube twice and left at RT
for 10 min for cross-linking. 2.5 ml of 2.5 M glycine was then added to the tube to stop cross-
linking. The content was quickly mixed by inverting the tube twice and left at RT for 5 min, and
then on ice for at least 15 min. Subsequently, cells were collected by centrifugation (2000 rpm,
10 min), and the cell pellet was frozen on dry ice and then stored at -80°C.
Modifications to the time course experiment

Auxin-induced protein depletion
Where required, auxin was added after 10 h 1NM-PP1 treatment and cells (SMC2-mAID-
GFP cells, CAP-H-mAID-GFP cells, CAP-H2-mAID-GFP cells) were further incubated for 3
hours to deplete AID-tagged proteins. Cells were then released from the G2 arrest as described
above, except that auxin was kept in the 1NM-PP1 washout media and in the media during all
further incubations. Note that the G2 cells for CAP-H-mAID-GFP and CAP-H2-mAID-GFP are
either GFP-negative (degron active) or GFP-positive (degron not activated), depending on auxin
addition (indicated in Tables S1, S2).
Cell sorting to isolate cells depleted of target proteins

As a control, in some cases (indicated as “sorted”) SMC2-AID-GFP cells were FACS-
sorted after cross-linking to exclude residual GFP-positive cells, which had not responded fully
to auxin. Some wild type cells were also sorted. Sorting did not alter the Hi-C data as compared
to unsorted cells, indicating that degron-mediated protein degradation was very efficient (Fig.
4A, Fig.S8 (set #4) and Fig.S20A (set #1).
Nocodazole addition to prevent anaphase entry

Nocodazole was added for 30 minutes prior to 1NM-PP1 washout only for the t ≥ 30 minute
time points (Replicate 1 of WT) or for t ≥ 60 minutes samples (all other time courses; Table S1).
Nocodazole was present in 1NM-PP1 washout media and in the media during further
incubations. No nocodazole was added for cells collected at any of the other time points.
Microscopy on fixed cells
Fixation of chromosomes from mitotic cells

Cells in media (200 µl) were directly fixed in 1 ml of ice-cold Methanol/Acetic acid
solution (3:1) for > 30 minutes. Fixed Cells were centrifuged at 2000 g for 1 min, applied on
slides, air dried, and stained with DAPI in Vectashield antifade mounting medium (H-1200,
Vectashield). Images were taken by DeltaVision microscopy.
Lamin B1 and Histone H3 Phos-Ser10 staining

Cells were fixed in pre-warmed 4% Formaldehyde/PBS for 10 min and permeabilized with
0.15% triton for 5 minutes. The following steps were then performed at RT. Cells were blocked
with 5% BSA/PBS for 30 minutes. Anti-lamin B1 mouse monoclonal antibody (1:100, Zymed)
and Anti-histone H3 phospho-Ser10 rabbit polyclonal antibody (1:500, Millipore) diluted in the
blocking buffer were applied to the cells for 1 hour. Cells were washed with PBS 3x for 5
minutes. Secondary antibodies (Molecular Probes, ThermoFisher Scientific) anti-mouse and anti-
rabbit coupled with Alexa Fluor 488 and 594, respectively and diluted 1:500 and 1:1000 in the
blocking buffer, were applied to the cells for 30 minutes. Cells were washed with PBS three
times for 5 minutes. DNA was stained with Hoechst 33452 and mounted with Prolong diamond
(Molecular Probes, ThermoFisher Scientific). Images were taken by DeltaVision microscopy.
DeltaVision microscopy
3D datasets were acquired using a cooled CCD camera (CoolSNAP HQ; Photometrics) on a
wide-field microscope (DeltaVision Spectris; Applied Precision) with a 100× NA 1.4 Plan
Apochromat lens. The datasets were deconvolved with softWoRx (Applied Precision), converted
to Quick Projections in softWoRx, exported as TIFF files, and imported into Adobe Photoshop
for final presentation.
Chromosome length measurements

Samples were prepared essentially by the same method used for Hi-C samples.
WT/CDK1as cells were treated with 1NMPP1 for 10 hours. After 1NM-PP1 washout, cells were
plated on poly-L-lysine coated coverslips and incubated with the corresponding media for either
30 or 60 minutes. Then the cells were rinsed with PBS and fixed with 1% formaldehyde in PBS.
Note: Nocodazole was added to the culture 30 minutes prior to 1NM-PP1 washout and kept in
the culture thereafter. MG132 was added to the culture at t = 30 minutes after release and cells
were fixed 30 minutes after that. Ten or more pictures were taken for each condition using the
Zeiss Airyscan microscope and analysed using IMARIS.
We measured the length and width of the longest chromosomes in DT40 cells fixed with
1% formaldehyde solution as used in the preparation of Hi-C specimens. It was not possible to
measure all chromosomes in cells fixed under these Hi-C conditions, so by choosing the longest
chromosome in each cell, we could be confident that we were measuring and comparing the
same chromosome (chromosome 1). In order to identify chromosome 1 in each mitotic cell, we
measured the length of several candidate chromosomes in each cell using IMARIS to track that
chromosome through the three-dimensional data set. The measurement reported was for the
longest chromosome. In order to calculate the chromosome width in each cell, avoiding
centromeres and telomeres, we measured the width of the longest chromosome at 5 different
positions.
Visualization of a repeating structure on chicken mitotic chromosomes

Asynchronously growing WT/CDK1as, SMC2-mAID-GFP/CDK1as, CAP-H-mAID-
GFP/CDK1as, CAP-H2-mAID-GFP/CDK1as, CAP-H-GFP/CDK1as cells were treated with
colcemid (100 ng/ml) for 2.5 hours. Cells were treated with 75 mM KCl for 5 minutes then fixed
with ice-cold Methanol/Acetic acid. Cells were spread on coverslips, mounted with vectashield
containing DAPI (Vectashield). 3D datasets were acquired using a cooled CCD camera
(CoolSNAP HQ; Photometrics) on a wide-field microscope (DeltaVision Spectris; Applied
Precision) with a 100× NA 1.4 Plan Apochromat lens. The datasets were deconvolved with
softWoRx (Applied Precision), converted to Quick Projections in softWoRx, exported as TIFF
files, and imported into Adobe Photoshop for final presentation.
This hypotonic treatment and spreading procedure caused a reorganization of the
chromosome structure that appears to reveal the underlying helical organization of the
chromosome scaffold. This helical organization is not seen in minimally perturbed chromosomes
fixed with formaldehyde, as used for our Hi-C studies, and where the chromatin distribution
appears to be uniform. In the chromosome spreads, we could unambiguously identify
chromosomes 1 and 2 and could count the number of gyres, and therefore calculate the amount
of DNA per gyre. This number is almost certainly an overestimate, because we did not see gyres
at centromeres or telomeres. For Chromosome 1 we counted 10.5 ± 3.8 gyres, yielding 18.7
MB/gyre (n = 19 chromosomes). For chromosome 2 we counted 8.5 ± 2.4 gyres, yielding 17.7
Mb/gyre (n = 17 chromosomes). We note that these chromosomes had been arrested in mitosis
for up to 2.5 hours with a colcemid block, so they would be expected to be more compacted than
the 60 minute time point analyzed by Hi-C.
Helical distribution of GFP signal in single sister chromatids

SMC2-mAID-GFP/CDK1as, CAP-H-mAID-GFP/CDK1as, CAP-H2-mAID-GFP/CDK1as,
CAP-H-GFP/CDK1as cells were treated with 2 µM 1NM-PP1 for 4h without auxin. Cells were
plated on poly-lysine coated coverslips immediately after 1NM-PP1 washout and incubated for
40 minutes. The cells were then fixed with 4% Formaldehyde/PBS for 10 minutes, permeabilized
with 0.15 % Triton/PBS for 5 minutes, stained with Hoechst solution, mounted with Prolong
Diamond antifadant and imaged using the Airyscan module on a Zeiss LSM 880 confocal, using
a x100 alpha Plan-Apochromat objective. Hoechst 33342 was detected using a 405 nm Diode
laser and a 420-480 nm bandpass emission filter, Alexa488 was detected using the 488 nm line
of an Argon laser and a 495-550 nm bandpass emission filter, Step size for Z stacks was set to
0.145 µm. 3D datasets were visualized and analyzed using Imaris V8.4 (Bitplane, Oxford
Instruments, Oxfordshire, UK). Images show single sections from three-dimensional data stacks
and are representative of two biological replicate experiments. Cells had been arrested in mitosis
for up to 2.5 hours with a colcemid block, so they would be expected to be more compacted than
the 60 minute time point analyzed by Hi-C.
Immunoblot analysis
Typically 0.5-1 x 106 cells were loaded in each lane of a 7.5 % SDS-PAGE gel. Proteins
were transferred to PVDF membranes at 200 mA for 2.5 hours in transfer buffer containing 119
mM Tris (pH 8.5), 40 mM glycine, 0.1% SDS, and 20% Methanol. Membranes were blocked
with 5% skimmed milk in PBS (154 mM NaCl, 1.5 mM KH2PO4, 5.5 mM Na2HPO4) for 30
minutes, then incubated with relevant primary antibodies recognizing α-tubulin (1:2000, DM1A,
Sigma-Aldrich; as a loading control), SMC2 (1:1000) (17), or GFP (1:1000) (gift from Simona
Saccani, University of Edinburgh) with 1% skimmed milk in PBS/tween20 (1%) for 3 hours up
to overnight. Membranes were washed three times for 5 minutes with PBS/tween20 (1%).
Membranes were incubated with IRDye-labeled secondary antibodies diluted in 1% skimmed
milk in PBS/tween20 (1%) (1:10,000) for 45 minutes followed by three 5 minute washes with
PBS/tween20 (1%). Membranes were kept in PBS until fluorescence intensities were determined
using a CCD scanner (Odyssey; LI-COR Biosciences) according to the manufacturer’s
instructions.
Flow cytometry analysis

DNA content: Cells were suspended overnight in ice-cold 70% ethanol. The next morning,
cells were rinsed with PBS then re-suspended in PBS containing 100 µg/ml RNase A and 5
µg/ml propidium iodide. Samples were then analyzed using a FACSCalibur flow cytometer
following the manufacturer’s instructions. Data was analyzed using FlowJo V10.3. WT cells
were gated for viability based on forward and side scatter (FSC/SSC), from which single cells
were selected based on FSC height (H) and width (W). These gates were applied to all samples
and DNA content was plotted as a histogram of FSC-H.
Chromatin Enrichment for Proteomics (ChEP)

Synchronized Cdk1as cells were crosslinked with 1% formaldehyde at specified time points
in mitosis and chromatin was extracted from the crosslinked cells by the ChEP protocol (58).
Briefly, 5 x 107 cells were fixed for 10 minutes by addition of formaldehyde to the cell culture.
Formaldehyde was quenched by 125 mM glycine for 5 minutes. Crosslinked cells were washed
with TBS (20 mM Tris pH 7.4, 150 mM NaCl) and frozen in liquid nitrogen. Cells were lysed on
ice in Lysis buffer (25 mM Tris pH 7.4, 0.1% Triton X-100, 85 mM KCl, protease inhibitors: 1
mM PMSF, 1 µg/ ml each of chymostatin, leupeptin, antipain, pepstatin A). Cells were washed
with Lysis buffer and the pellet was resuspended in 300 µl SDS buffer (50 mM Tris pH 7.4, 10
mM EDTA, 4% SDS, protease inhibitors as above) for 10 minutes at room temperature. Next, 3
volumes of Urea buffer (10 mM Tris pH 7.4, 1 mM EDTA, 8M Urea) were added and samples
centrifuged at 16,000 x g for 30 minutes. The pellet was again resuspended in 1 volume of SDS
buffer, then 3 volumes of Urea buffer was added before centrifugation again. The pellet was
washed with SDS buffer before resuspension in ice cold storage buffer (10 mM Tris pH 7.4, 1
mM EDTA, 25 mM NaCl, 10% glycerol). Chromatin DNA was sheared by sonication. The
solubilized chromatin was mixed with 3x SDS sample buffer (6% SDS, 150 mM Tris pH 6.8,
30% glycerol) and heated at 95°C for 30 minutes to reverse the crosslinks. 10% of the sample
was electrophoresed in a NuPAGE Bis-Tris gel (NP0335, Invitrogen). The de-crosslinked
chromatin proteins were in-gel digested with 2µg trypsin overnight (> 16hours). The tryptic
peptides were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-
MS/MS). Data was analyzed with MaxQuant 1.5.7.4 (85). The abundance of proteins was
estimated using the iBAQ algorithm (86), and normalized by the copy number of histone H4
(87).
ChIP-seq data re-analysis.

We used the publicly available CAPH ChIP-seq dataset obtained in DT40 cells
(GSE45552). We processed the raw sequences following the steps of the ChIP-seq pipeline of
the ENCODE consortium (88), available at https://github.com/ENCODE-DCC/chip-seq-
pipeline/ .
Hi-C protocol
Chromosome conformation capture was performed as described previously (89). Briefly, 10-
20x106 cells were cross-linked in 1% formaldehyde for 10 minutes and quenched in 125 mM
glycine. Cells were snap-frozen and stored at -80°C before cell lysis.
Cells were lysed for 15 minutes in ice cold lysis buffer (10 mM Tris-HCl pH8.0, 10 mM
NaCl, 0.2% Igepal CA-630) in the presence of Halt protease inhibitors (Thermo Fisher, 78429)
and cells were disrupted by homogenization with pestle A for 2x 30 strokes. Chromatin was
solubilized in 0.1% SDS at 65°C for 10 minutes, quenched by 1% Triton X-100 (Sigma, 93443)
and chromatin was digested with 400 units of HindIII (NEB, R0104) overnight at 37°C.
Fill-in of digested overhangs by DNA polymerase I, large Klenow fragment (NEB, M0210)
in the presence of 250 nM biotin-14-dCTP (Life Technologies, 19518-018) for a minimum of 90
minutes was performed prior to 1% SDS based enzyme inactivation and dilute ligation with T4
DNA ligase (Life Technologies, 15224) for 4 hours at 16°C. Cross-links of ligated chromatin
were reversed overnight by proteinase K (Life Technologies, 25530-031) incubation at 65°C.
DNA was isolated with 1:1 phenol:chloroform, followed by 30 minutes of RNase A incubation.
Biotin was removed from unligated ends by incubation with 15 units of T4 DNA
polymerase in the presence of 25 nM dATP/dGTP. DNA was sheared using an E220 evolution
sonicator (Covaris) and size selected with Agencourt AMPure® XP (Beckman Coulter) to 150-
350 bps. After end repair in a mixture of T4 polynucleotide kinase (25 units; NEB, M0201), T4
DNA polymerase (7.5 units; NEB, M0203L) and DNA polymerase I, large (Klenow) fragment
(2.5 units; NEB, M0210) at 20°C for 30 minutes, dATP was added to blunted ends using 15 units
of polymerase I, large fragment (Klenow 3’ → 5’ exo-) (NEB, M0212L) at 37°C for 30 minutes.
Biotinylated DNA was collected by incubation in the presence of 10 µl of streptavidin
coated myOne C1 beads (Life technologies, 650.01) and Illumina paired-end adapters were
added by ligation with T4 DNA ligase (Life technologies) for 2 hours at room temperature. A
PCR titration (primers PE1.0 and PE2.0) was performed prior to a production PCR to determine
the minimal number of PCR cycles needed to generate a Hi-C library. Primers were separated
from the library using Ampure size selection prior to 50 bp paired-end sequencing on an Illumina
HiSeq sequencer (Life Technologies).
Hi-C data analysis

Briefly, we processed Hi-C data as described in (36), with multiple modifications, including
using the galGal5 genome assembly. All scripts mentioned below to be from the Dekker Lab c-
world pipeline are available at a GitHub repository (90). cMapping at
https://github.com/dekkerlab/cMapping; iterative correction (balancing) at
https://github.com/dekkerlab/balance and analysis scripts at
https://github.com/dekkerlab/cworld-dekker. The Mirny Lab software, including the cooler
library for storage and analysis of Hi-C data is available at https://github.com/mirnylab.
Mapping sequenced Hi-C reads

Fastq files were mapped and binned using the c-world mapping pipeline. Briefly, 50 bp
paired end reads obtained as fastq files were truncated to 25 bp starting at the 5’ end. Then, they
were iteratively mapped (33) to the Red Jungle Fowl laboratory line chicken genome (UCSC
Genome Browser assembly ID: galGal5, Dec. 2015, Accession ID: GCF_000002315.4; NCBI
genome/111 (Gallus gallus)(91). Uniquely mapped, paired reads were kept and assigned to a
HindIII restriction fragment assigned through its 5’ position. Mapped reads were filtered for
same fragment ends and uniqueness, excluding PCR duplicates (defined as sequence matches
with the exact same start and end).
Hi-C based karyotyping

Genome wide interaction matrices from Hi-C were binned at 1 Megabase resolution. For
each bin, we summed up the interactions of this bin with all other bins. Chromosome
normalization was obtained by dividing these sums with the total number of interactions per
chromosome, corrected for the chromosome size (in Megabases). Genome wide normalization
was obtained by dividing each bin sum by the genome wide number of counts (matrix total). The
difference between sums normalized by chromosome and matrix indicates copy number
alterations and was displayed for each chromosome on a log2 scale.
Contact probability (P(s)) curves

We used the mapped reads to calculate the functions of contact frequency P(s) vs genomic
separation s. We split all genomic distances between 1 kb and 1Gb into bins of exponentially
increasing widths, such that the upper edge of every bin was 1.12 larger than the lower edge. For
every such separation bin, we found the number of observed cis-interactions within this range of
separations and divided it by the number of all loci pairs, separated by such distances in cis.
Binning and balancing of Hi-C data

Unique valid pair reads were binned to 10kb, 50 and 100 kb bins, using cooler package, and
noisy or low signal bins were excluded prior to balancing using the MADmax filter: we
removed all bins, whose coverage was 7 genome-wide median deviations below the median bin
coverage. Matrices were balanced by iterative correction (IC), equalizing the sum of every
row/column to 1.0 (33), using cooler (https://github.com/mirnylab/cooler).
Compartment analysis
Compartments were quantified using principal component analysis on 100 kb binned data
using the matrix2compartment perl script in our c-world pipeline. The largest eigenvector (eig1)
typically represents the compartment profile(3, 32, 92). When compartment signals were low or
absent, we checked consecutive eigenvectors to confirm loss of compartments. Per convention
A/B-compartments were assigned by gene density, so that the A-compartment was more gene-
dense than the B-compartment.
In order to measure the strength of compartmentalization, we used the observed/expected
Hi-C maps, which we calculated from 100 kb iteratively corrected interaction maps of cis-
interactions by dividing each diagonal of a matrix by its chromosome-wide average value. In
each observed/expected map, we rearranged the rows and the columns in the order of increasing
eigenvector value. Finally, we aggregated the rows and the columns of the resulting matrix into
30 equally sized aggregated bins, thus obtaining a compartmentalization plot (“saddle plot”).
Insulation analysis (TADs)

TAD calling was performed on 50 kb binned data exactly as described previously (32). We
first determined the optimal insulation square for TAD calling with our c-world pipeline perl
script matrix2insulationRange. We used a range of insulation squares from 50 kbp (istart
variable) to 1 Mb (iend variable) with a 50 kb (bin size) step (istep variable) to sweep through
the insulation topology. From this sweep, we determined to use a 250x250 kb sliding square (is =
250000) along the matrix diagonal without smoothing (ss = 50000) for capturing the aggregate
TAD signal for galGal5 (Fig. S5). For 40 kb binned HelaS3 (ATCC CCL2.2) Hi-C data, we
detected TADs using a 520x520 kb sliding square (is = 520000) without smoothing (ss = 40000).
These settings were used as input variables for insulation square analysis with the
matrix2insulation perl script from our c-world pipeline. Briefly, the inter quartile (IQR) mean
signal within the square (im = iqrMean) was assigned to the diagonal bin and this process was
repeated for all bins. The insulation score was then normalized relative to average score of the
insulation scores across each chromosome by calculating the log2 ratio of each bin’s insulation
score and the mean of all insulation scores, as described previously (32). Minima along the
normalized insulation score vector represent loci of high local insulation and are interpreted as
TAD boundaries. For aggregate insulation score pileups, bed files with TAD boundary positions
from G2 arrested cells were used as input for each mutant cell line separately (ebf variable).
TAD boundary pileups were generated for 50 kb steps (mindist = 50000) covering 500 kb
upstream and 500 kb downstream from TAD boundaries (ezs = 500000) with a maximum
interaction distance of 500 kb (maxdist = 500000) using our elementPileUp c-world perl script.
Coarse-grained model of contact probability decay in mitotic chromosomes
In the coarse-grained model loops are regularly placed along the axis of the chromosome (z-
axis) with a spacing Δz. Each loop is represented by a cylindrical wedge, where loci that belong
to this loop are distributed normally along the z directions, with mean at the base of the loop and
the standard deviation σ), and normally in the angular position with the mean at the orientation
of the loop and the standard deviation φ. Orientation of each loop depends on the model and can
be uniformly random (Randomly oriented uncorrelated loops), correlated with its neighboring
loops, forming an angular random walk (Loops with correlated orientation), or form a random
walk while having a preferred orientation (Loops with spiral staircase orientation).
Below, we derive an approximate expression for the probability of a contact between two
loci Pc(s), separated by genomic distance s, in a chromosome compacted into an array of
consecutive loops. Naturally, this expression has two regimes: (a) the intra-loop regime at shorter
genomic separations, where the two loci are located on the same loop, and (b) the between-loop
regime at larger separations, when loci are located on different loops. If the lengths of loops are
random and distributed exponentially, the transition between the regimes occurs around the
genomic separation comparable to the average loop length lavg:
(1)
Contacts within loops

The simplest model of individual loop conformations is a random walk in 3D. In this case,
the contact probability decays as a power-law with a -3/2 exponent, assuming poor solvent
conditions in the melt of loops, and neglecting correction for the closed loop:
(2)
This scaling is consistent with Pc(s) observed even at larger distances during interphase in
very large oocyte nuclei (93). Below, we will not attempt to improve this simple model, and
instead focus on long-distance behavior of Pc(s), which reflects the mutual arrangement of loops.
Contacts between loops

By definition, a contact between two loci occurs when they have similar coordinates, either
in Cartesian or cylindrical coordinates:
For two loci located on different loops, we can simplify this expression drastically by
assuming that the probabilities of overlap at each coordinate are independent of each other:
(3)
Randomly oriented uncorrelated loops

In order to estimate the probability of contact between loci on different loops, we have to
make extra assumptions on the mutual orientations of these loops. In the simplest case, each loop
is randomly oriented and the orientations of consecutive loops are not correlated with each other.
In this case, both and are independent of the separation between the
loops and thus integrate to a constant, leaving:
We can calculate this remaining expression easily if we assume that the loci within each
loop are normally distributed along the z-axis, each around the base of its loop:
In this expression, N(x, mean, s.d.) is the PDF of the normal distribution, i and j are the
genomic coordinates of the two loci, zi and zj are the axial coordinates in the 3D genomic
structure, and are the expected axial coordinates of the i-th and j-th loci (which, by our
assumption, are equal to the axial coordinates of their corresponding loop bases), σ is the
measure of the spread of loop along the axis of the chromosome, i.e. the high of each loop, and
is the delta function that limits the integral only to the conformations where the
particles have the same axial coordinate. Taking this integral gives us:
Finally, if the loops are regularly places along the axis of the chromosome with a spacing
Δz, than the expected axial distance between the bases of two loops is equal the expected number
of loops between these two loci, multiplied by Δz:
(4)
PcZ(s) describes the decay of contact probability due to the decreasing axial overlap of more
distant loops and, as illustrated in Fig. 2B, produces a characteristic drop-off in log-log
representation of Pc(s). The drop takes place at when the number of loop s/lavr exceeds
~2 /Δz the ratio of the loop high and the separation between loop.
For example, at prophase a drop at 4.5Mb for loops of the average size lavr = 80Kb requires
56 loops per layer. Hence 2 /Δz = 56; then for = 200nm of loop high, one gets Δz=7.5nm
separation between neighboring loops. This is a tight packing at the scaffold, but feasible with
10nm fiber emanating from condensins, possibly requiring the scaffold to wiggle inside the
chromosome. For prometaphase, a drop at 10Mb and 80Kb loops leads to 125 loops per layer,
and the spacing of 3nm, which is smaller than protein domains of condensin, necessitating nested
loops and considerable winding of the scaffold inside the chromosome.
Loops with correlated orientation

Pcz(s) alone does not fully explain the experimentally observed Pc(s) curves: it explains the
origin of a drop in contact probability at longer distances, but does not explain the origin of a
gradual decay before the drop. Equation (3) then suggests that this extra gradual decay might be
due to the angular overlap . Specifically, if the angular orientations of consecutive
loops are somehow correlated, forming the angular (1D) random walk, this would increase the
frequency of angular overlaps for loci in adjacent loops and make it smaller for loci that are
contained in distant loops.
(5)
We can derive the frequency of angular overlaps explicitly:
Here, θi and θj are the angular coordinates of the two loci, which are distributed normally
around the mean angular orientation of their loops (0 and , correspondingly) with the standard
deviation φ. The correlations in the orientations of adjacent loops lead to the fact that the mean
angular orientation of the loop with the j-th locus, is normally distributed itself around the
mean of 0 (which we picked to be the angular orientation of the loop with the i-th locus, without
a loss of generality) and the standard deviation of . The latter is calculated from the
assumption the angles of two consecutive loops on average differ by ; thus, between the i-th
and j-th locus there are s/lavg loops, each taking a random step of , thus producing a 1D random
walk with the standard deviation of . Taking all integrals gives us:
(6)
Pcang,corr describes the decay of contact probability with distance due to gradually decaying
correlations of more distant loops. For a range of parameters, it can produce a characteristic -0.5
power-law decay of Pc(s), before the random walk fills the whole In combination with the
z
drop-off decay due to decreasing axial co-localization, Pc , these two terms produce a prophase-
like three-regime decay of contact probability, as illustrated in Fig. 2B.
Interestingly, when the angular random walk fills a full circle the angular part of the contact
probability becomes constant and independent of s, i.e. Pcang corr(s)~s-0.5, while the angular
displacement <2 For more loops per layer Pc(s)=const is expected to appear. Since
we don’t see this regime in the data, it means that <2 for s within a layer. Hence for a
drop at 10Mb and 80Kb loops, we get 125 loops per layer and , so the angle between
loops should be below or around 30 degrees.
Loops with spiral staircase orientation

Finally, we build a model that could explain the non-monotonic dependency of contact
probability with separation, observed in Hi-C of prometaphase chromosomes. This non-
monotonicity could be explained by spiralization of chromosomes, previously observed with an
electron microscope. In our model, we can describe this spiralization as periodicity of the angular
orientations of loops. In order to agree with the experimental data, we have, however, to combine
this periodicity at larger scales with random correlated angular loop orientations at shorter
separations.
These properties could all be achieved when the angular orientations of loops follow a 1D
random walk under the constraint that each loop cannot deviate too much from its preferred
orientation, set by the spiral, i.e., linearly proportional to its index. The probability of an angular
overlap between loci on two loops can be calculated as the probability of a return of such 1D
random walk to its initial position after s/l_avg steps. These random walks are known under the
name of the Ornstein-Uhlenbeck (OU) processes, and their properties, including the return
probability, have been studied in the theory of stochastic processes (94):
Here, Δθ is the systematic shift of the angle per loop (which produces the spiral phenotype).
nreturn is the parameter of the Ornstein-Uhlenbeck process characterizing the variability of the
angular loop orientations around their optimal orientation: the loops deviate on average by
from their optimal angular orientation and the nreturn consecutive loops have correlated
orientations. We also modified the standard formula for the return probability of the trending OU
process to account for the fact that, in angular coordinates, two points overlap when they have
the same angle modulo 2*pi.
Bridging the in-loop and between-loop regimes
Note that both expressions of Pcin and Pcbetween contain arbitrarily defined coefficients Cin
and Cbetween. In order to obtain a unified expression that describes the contact probability at all
length scales, we need adjust these coefficients, such that Pcin(s) and Pcbetween could be directly
compared to each other. Both of these coefficients are arbitrary and their ratio only affects the
narrow transitional region at s ~ 1-3 lavg. In the calculations presented in the paper, we calculate
these coefficients by matching the two curves at the separation of two average loop sizes:
Polymer models of mitotic chromosomes

The design of Langevin molecular dynamics simulations of chromosomes
In order to test the proposed architectures of prophase and prometaphase chromosomes
against our Hi-C data, we simulate polymer models of chromosomes using Langevin molecular
dynamics. The overall design of these simulations is similar to that in (8, 24), with a few major
differences. We model 10nm ‘beads-on-string’ chromatin fiber of nucleosomes as a chain of
10nm particles, each representing one nucleosome with 200bp of DNA. These particles are
connected by springs with a using a harmonic potential with an equilibrium length of 10nm and
the stiffness coefficient of 1 kbT/nm2. We simulated repulsion between spatially overlapping
nucleosomes using the following force potential:
This potential is designed to be constant of 5.0 kbT until r = 7-8nm, and then rapidly go to
zero around 10.5 nm. The limited maximum overlap energy of 5.0 kbT allowed chromatin fibers
to pass through each other occasionally and thus accounted for the strand-passing activity
topoisomerase II. In all of our simulations, we additionally compact chromosomes using
cylindrical constraints to impose the high chromatin density of one nucleosome per 11nm x
11nm x 11nm box, as observed in EM images of mitotic chromosomes. The particular geometric
parameters of the cylindrical constraint varied between simulations. We imposed this constraint
using a harmonically increasing potential with k=0.1 kbT when monomer crossed the boundaries
of the constraining cylinder. We perform Langevin dynamics polymer simulations using
OpenMM, a high-performance GPU assisted molecular dynamics API (95, 96). We used the
following parameters of the Langevin dynamics – variable time step to achieve the relative
accuracy of 0.001, the friction coefficient of 0.01, particle mass of 1.0 and the temperature of
300K. We ran the simulations until the simulated P(s) curves (see below) stopped changing at
logarithmic time scales, which occurred after 1.5e7 timesteps in prophase simulations and after
3e6 timesteps in prometaphase simulations. We visualized the resulting chromosome structures
using Pymol (DeLano, Warren Lyford. "PyMOL." (2002)).
Simulations of prophase and CAP-H2-depleted chromosomes
We simulated prophase chromosomes as arrays of consecutive loops compacted into a
cylindrical shape with a high chromatin density. As in (8), we first randomly selected a subset of
particles to be bases of consecutive loops, such that the loop lengths were exponentially
distributed with an average of lavg. We modelled loop-forming condensins by imposing extra
harmonic bonds between the particles representing loop bases. Finally, we tethered the first and
the last particles of the chromosome to the ends of the compacting cylinder using a harmonic
attraction potential along z-coordinate with k=0.15 kbT/nm2. We equilibrated the each
simulation for 1.5e7 steps to allow for slow large-scale rearrangement of the flexible
chromosome scaffold. To match the simulations with the WT prophase and CAP-H2
prometaphase experiments, we systematically varied the two model parameters: (1) the average
loop length, lavg, from 20 kb and 100 kb, with a step of 10 kb and (2) the average linear density
of loops along the axis of the constraining cylinder, Nloops/L, from 50 loops/μm to 350 loops/μm,
with a step of 50 loops/μm. For each parameter set, we modeled a chromosome containing 500
average loop lengths.
Simulations of prometaphase and CAP-H-depleted chromosomes

We simulated prometaphase chromosomes as helical arrays of consecutive nested loops
compacted into a cylindrical shape with a high chromatin density. As in the prophase simulations
above, we first imposed an array of consecutive loops with the average loop length of lavgouter.
To account for the action of condensin I, we further split each of these “outer” loops into an
array of smaller exponentially distributed “inner” loops, with an average length lavginner. To
test if the Hi-C data is consistent with helical winding of prometaphase chromosomes, we fixed
the bases of the “outer” loops along a 3-dimensional helical path winding around the axis of the
constraining cylinder. We imposed this helical path by tethering each loop base to its specific
position along the helix with a harmonic potential with k=0.04 kbT/nm2. We equilibrated each
simulation only for 1e6 steps, since the motion of the backbone was suppressed and only
individual loops had to equilibrate. To match the simulations with the prometaphase
experiments, we varied four parameters of the model: (1) the average inner loop size, lavginner,
from 20 kb to 100 kb every 20 kb; (2) the average outer loop size lavgouter as a multiple of the
average inner loop size lavginner, from 1 to 11 inner loops, with a step of 2; (3) the average
amount of DNA per helical turn, P, from 2 to 15 Mb, with a step of 1 Mb; (4) the pitch of the
helical scaffold, b, from 50 to 300nm, with a step of 50nm. As above, for each parameter set, we
modeled a chromosome containing 500 lavgouter. For each parameter set, the length of the
constraining cylinder matched the axial length of the helical scaffold, and the radius of the
cylinder was adjusted to reach the density of 1 particle per 11nm x 11 nm x 11 nm box.
Simulated Hi-C and P(s) calculation for polymer models

We then used our polymer models to perform in-silico Hi-C. For each polymer model, we
picked the chromosome structure at the end of the simulation and found all pairs of contacting
monomers, i.e. pairs of monomers separated by less than 51nm in 3D space. As in the
experimental Hi-C, we used these pairs to calculate the contact frequency vs separation P(s)
curves.
Model selection via comparison of simulated and experimental P(s) curves

To find which simulations produce the best agreement with the experimental data, we
systematically tried all possible combination of the model parameters. For each parameter set,
we performed a simulation, generated a P(s) curve and calculated the root mean square (r.m.s.)
difference with the experimental P(s) in the log10-log10 scale, after normalizing both of the
curves at the specific distance (see the table below). Our simulations cannot predict P(s) both at
very short separations, below a loop size, and at very long separations, after the drop-off; for that
reason, we limit our P(s) discrepancy computations to ranges shown in the table below. Finally,
we equalize the contribution of the short- and long-distance parts of the P(s) (Table S4) by
calculating the r.m.s. P(s) difference in each of these regions separately and reporting the mean
of the two.
P(s) estimation for prophase models with sister chromatid cohesion

In order to understand how the presence of a spatially proximal sister chromatid could
change the P(s) curves we performed a mock-up simulation. We considered the 3D structure of
the best-fitting prophase model and added to it its identical copy shifted along the direction
perpendicular to the chromatid axis by distance Δxsister. We then calculated the P(s) for this
structure, under the assumption that our in-silico Hi-C procedure could not tell apart the
corresponding identical loci on the two chromatids. We then repeated this procedure for different
values of Δxsister, from 0 (a complete overlap between the two chromatids) to 2 chromatid radii (a
complete segregation of the chromatids).
Estimation of the rate of loop formation at condensin binding sites.

We calculated the average contact frequency around condensin binding sites at 10kb
resolution, considering interactions formed by loci located up to 1Mb away from the condensin
sites. We then normalized these average maps by the distance-dependent decay of contact
frequency P(s), thus obtaining average “observed-over-expected” maps of contact enrichments
around condensin binding sites.
We used the simulated 3D models of prometaphase chromosomes at 60 minutes to calculate
the average contact map around a loop anchor. We simulated a Hi-C map for the whole model at
10kb resolution and averaged its segments around 1000 randomly chosen loop anchors. As with
the experimental maps, we then normalized it by the decay of contact frequency with distance
from the same model.
The average map of contacts formed by loop anchors in our 60 minute model showed a
“plus-sing” pattern, corresponding to the enrichment of contacts between loop anchors and their
neighboring loci. In our experimental Hi-C data, the condensin binding sites displayed a similar
contact pattern, albeit noticeably weaker, suggesting that condensin binding sites harbor a loop
anchor only in a fraction of sampled cells.
We then quantified the strength of these “plus-sign” patterns in our experimental and
simulated maps as the median observed-over-expected enrichment of contacts formed between
the sites in question (condensin binding sites, for experimental data, and a loop anchor, for
simulated data) and their neighboring loci, divided by the median observed-over-expected of
contacts between all pairs of neighboring loci. Comparing the strengths of “plus-signs” at
condensin sites to the strength of such patterns at loop anchors in our simulations allowed us to
estimate how often condensin sites harbor a loop in the sampled population of cells.
Simulations of nested loop formation by loop extrusion.

To test if the process of loop extrusion can form nested loops, we performed lattice loop
extrusion simulations as previously described in (25). Briefly, such simulations aim to model the
positions and sizes of loops produced through the process of loop extrusion and do not explicitly
consider the 3D chromosome conformation. In these simulations, a chromosome is divided into a
1D lattice of sites and the condensin-extruded loops are represented by a pair of sites, loop
anchors, which are brought together at the base of a loop. We simulate loop extrusion by step-
wise shifting of the two loop anchors of each condensin away from each other over time. To
simulate the chromatin binding/unbinding dynamics of condensins, we occasionally remove
them from the lattice and put back to a randomly chosen location, where they start a new loop.
The code for these simulations is available at https://github.com/golobor/looplib/.
To simulate extrusion of loops by condensins I and II, we considered 20Mb of chromatin

fiber, divided into a lattice of 100,000 sites of 200bp each. We initialized the system by
randomly placing condensins I (one per 40kb) and II (one per 160kb) along the lattice, such that
the two loop anchors of each condensin were located on the adjacent lattice sites (i.e. they have
not yet extruded any loop). We then simulated the process of loop extrusion at the average speed
of 10 sites per second at each loop anchor. The average residence time was 2 minutes for
condensins I and 120 minutes for condensins II. We ran simulations for 2 hours of experimental
time.
Simulations of suppression of TADs and compartments by loop extrusion in prophase

Polymer simulations of an 8.3 Mb fiber with loop extrusion and compartmentalization are
performed as described in (24, 97) We chose parameters to reproduce loop length estimates in
interphase / 5min prophase / 7.5min prophase: Loop extrusion factor (LEF)
processivity/separation was 250kb/500kb = 1/2 (interphase), 125kb/31.3kb = 4 (5 min prophase),
250kb/25kb = 10 (7.5 min prophase). Other simulation parameters are: coarse graining =
400bp/monomer, LEF speed = 580 bp/s on each LEF side, boundary stopping probability=90%,
monomer volume density=0.15 (bead diameter)^3, compartmental interaction parameter=0.12
kT, repulsive core strength=3 kT, mean distance between CTCF sites=430 kb, crosslinking
radius for simulated Hi-C = 4 bead diameters. Hi-C maps, insulation tracks, Eigenvectors, and
insulation pileups are binned at 50 kb, compartmentalization plots at 100 kb. Example
configurations are colored linearly along the fiber from red to blue.
Figures S1-S27
Fig.S1
A DT40 karyotype Chromosomes galgal5
WT
SMC2-mAID
CAP-H-mAID
CAP-H2-mAID
1 2 3 4 5 6 7
-1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1
8 9 10 11 12 13 14
-1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1
21 22
15 17 18 19 20
-1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1
23 24 25 26 27 28 W
Z
-1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0.5 1 -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1
log2 normalied counts
B Integration sites C Translocations

200
1 2 3 7
180 0 CDKN1B
MIRLET7I
CDKN1B
160
140
110
120
Size (Mb)
100 7
80 63 SOX5 68.5 107 MIR221 113 137 MYC 143
60
All
All x
40
20 14
1
0
20
23
24
14 20 23 24
Fig.S1
Hi-C based karyotyping of cell lines. (A) Karyotype plots derived from 1Mb binned Hi-C
matrices. The sum of interactions for each bin with every other bin is normalized both genome
wide (gray lines) and by chromosome (colored by cell line) and displayed on the x-axis (log2
normalized counts) for each individual chromosome (y-axis) from Hi-C data obtained from cells
at t = 10 minutes (wild-type (black), SMC2-mAID (magenta), CAP-H2-mAID (red) and CAP-H-
mAID (blue)). A relative increase in chromosome normalized counts in comparison to genome
wide normalized counts indicates copy number gains, whereas a decrease shows copy number
loss. (B) Integration sites. Translocations are visible from Hi-C maps as an increased normalized
read counts at the intersection of the original locus and the site of translocation. The integration
of SOX5 near the MYC gene occurred at the generation of the DT40 cells line and has been
described previously (98). Our Hi-C maps show that MYC, SOX5 and MIR221 have
translocated to a single integration site on chromosome 1 (shown in green box), just upstream of
MIRLET7I (blue). (C) Translocations. Translocations show as an increase in Hi-C signal
between different chromosomes. We observed translocations between chromosomes 14 and 24
as well as 20 and 23 (dotted boxes), but not for the other chromosomes including chromosome 7
(green), which was used as our model chromosome throughout this manuscript.
Fig.S2
FACS analyis after release from synchronization
1.0K 1.0K 250
800 800 200
FL2-W
Count
SSC-H
600 600 150
400 400 100

95.6 85.4
200 200 50
0 0 0 2N 4N
0 200 400 600 800 1.0K 0 200 400 600 800 1.0K 0 200 400
FSC-H FL2-H DNA content
WT SMC2-mAID CAP-H2-mAID CAP-H-mAID
2N 4N 2N 4N 2N 4N 2N 4N
10h
1NM-PP1 7142 7755 7811
8282 7663
+0.50
Cell coount per timepoint
7696 8756 8854

+0.75
7441 7159 8151 8478
+1.0N
7314 6734 7341
+1.00
5941 7191 4628 5576
+1.50
5779 5854 5458 5712

+3.00
0 200 400 0 200 400 0 200 400 0 200 400

DNA content DNA content DNA content DNA content
Fig.S2
FACS analysis for synchronized cell cultures. FACS is shown for auxin induced and CDK1as
synchronized (10 hr 1NM-PP1) cell cultures. Gating was applied for live cells (top-left) and
singlets (top-middle) and gated cells were plotted as a histogram for DNA content (top-right).
The bottom plot shows the DNA content (propidium iodide) on the x-axis and time after 1NM-
PP1 release (in hours after release from G2 arrest) on the y-axis (top to bottom) for each auxin
induced cell line. Cells were not blocked at prometaphase with nocodazole except for the
samples indicated with red lines (1.0N). This analysis allows determination of the timing for exit
from mitosis as a loss of cells in G2/M (4N peak) and a subsequent increase in cells in the G2
peak (2N).
Fig.S3
NEBD visualized by Lamin staining
G2 7.5 min 10 min

Lamins in cytoplasm
merged
LaminB1
DNA
Ser10-P
2 µm
Interphase Prophase Prometaphase

Fig.S3
Nuclear envelope breakdown (NEBD) in CDK1as synchronized DT40 cells determined by
Lamin B1 staining. Lamin disassembly and chromosome condensation gradually occur at nuclear
breakdown. Lamin B1 localizes at the nuclear periphery (green pseudocolor) before and
throughout the cytoplasm after NEBD. Serine 10 of histone H3 becomes phosphorylated during
mitosis (H3K10ph, red). DNA stained by DAPI is shown in blue (pseudocolored).
Fig.S4
A Images corresponding to samples for condensin ChEP data
WT
3 x wash
G2 0 min 2.5 min 7.5 min 12 min
17 min 22 min 27 min 42 min 57 min 72 min
ChEP data
B WT C CAP-H-mAID D Cohesin/CTCF
0.02 NEBD NEBD NEBD SMC2-mAID WT

SMC2 CAPD2
SMC4 CAPG1 SMC1
CAPD2 CAPH SMC3
0.015 CAPG1 CAPD3 CTCF
CAPH CAPG2
CAPH2
0.010
Relative copy number /Histone H4
0.005
0.02 10
CAPD3 ratio CAPH/CAPH2 G2 0 5 10 15 20 25 30 35 40 45
CAPG2 Exp 1
CAPH2 8 Exp 2
0.015 SMC1 Exp 3
SMC3 AID-CAPH
6
0.010
4
0.005
2
0 0
Time (min) G2 0 5 10 15 20 25 30 35 40 45 G2 0 5 10 15 20 25 30 35 40 45
Fig.S4
Chromatin enriched proteomics (ChEP) data for CDK1as synchronized DT40 cells. Chromatin
enrichment for proteomics (ChEP) (58) identifies proteins associated with the DNA (see
Supplementary Methods). (A) Microscopy (DAPI staining of chromosomes) of cells at different
time points in mitosis. (B) The relative quantitation of chromatin bound condensin I, II and
cohesin subunits in CDK1as synchronized cells through WT prophase and prometaphase.
Numbers on the y-axis are arbitrary MaxQuant units (in millions), normalized for Histone H4
levels. Timing was made comparable to samples used for microscopy and Hi-C libraries based
on nuclear and chromosomal morphology in (A). (C) ChEP from CAP-H depleted (CAP-H-
mAID) cells indicates a loss of chromatin bound condensin I subunits after auxin mediated
depletion (top). The bottom plot shows the ratio of chromatin binding for CAP-H over CAP-H2
subunits after CAP-H depletion (dotted line) in comparison to 3 replicate control synchronized
time course experiments wherein CAP-H was not depleted. (D) ChEP for cohesin and CTCF
binding to chromatin in SMC2-depleted cells (auxin induced SMC2-mAID) and condensin
containing (un-induced) cells. Condensin depletion results in delayed loss of cohesin and CTCF
(indicated by the horizontal arrow) and retention of more cohesin and CTCF binding (indicated
by the vertical arrow).
Fig.S5
A Insulation analysis at varying length scales B Insulation score variance
WT-G2-S2-R3
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16
log
G2
0 0
normalized reads insulation score

2.5
500 kb 5
7.5
250 kb
100 kb 10
0.8
0
85
100 kb
250 kb
500 kb
Fig.S5
Insulation parameter settings for TAD analysis. (A) Insulation at different length scales. Hi-C
matrices were subjected to insulation analysis (depicted in interaction heatmap below). An
insulation score is calculated for each bin by summing all interactions occurring between loci
located up to a defined distance upstream and downstream of the bin (indicated by the diamond
squares in the bottom panel). This method is described in detail in (32). Bins with high insulation
(at a TAD boundary) have a low insulation score, as measured by fewer interactions occurring
across it. Bins with low insulation or boundary activity (the middle of a TAD) have a high
insulation score. Minima along the insulation profile are potential TAD boundaries. We
determined the optimal square size from a range of 50kb to 1Mb to be 250 kb. The large black
dotted square (chromatin domain) contains 2 smaller domains structures, indicated by green
squares. The specific insulation analyses using 100, 250 and 500 kb windows below the
interaction heatmap reveal that small insulation square sizes (<100kb) pick up small scale
fluctuations in insulation, whereas larger squares (>500 kb) miss weaker boundaries (green
dotted line derived from square above). The line graph in the bottom shows the fluctuation in
insulation score for each tested square size (100, 250, 500kb) for the same chromosomal region
as the interaction maps above. (B) Insulation score variance calculated with a square size of 250
kb for Hi-C data obtained at different times during prophase. The loss of variance in insulation
score as prophase progresses indicates loss of TAD structure (8).
Fig.S6
Contact frequency P(s) plots for all individual chromosome arms
WT-log-S1-R1 WT-G2-S1-R1
2
10
log G2 WT
contact frequency
1
10
0
10
Set #1
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10
Separation (bp) Separation (bp)
WT-G2-S1-R2 WT-10min-S1-R1 WT-30min-S1-R1 WT-30min-S1-R2 WT-60min-S1-R1

2
10
G2 10min 30min 30min 60min
contact frequency
1
10
0
10
Set #1
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Separation (bp) Separation (bp) Separation (bp) Separation (bp) Separation (bp)

2
10
contact frequency
1
10
0
10
Set #2
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
WT-0min-S3-R1 WT-2.5min-S3-R1 WT-5min-S3-R2 WT-7.5min-S3-R1 WT-10min-S3-R1

2
10
0min 2.5min 5min 7.5min 10min
contact frequency
1
10
0
10
Set #3
-1
10
-2
10
-3
10
-4
10
3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
WT-G2-S4-R4 WT-15min-S4-R2 WT-30min-S4-R4 WT-60min-S4-R3

2
10 G2 15min 30min 60min
contact frequency
Set #4 (GFP sorted)
1
10
0
10
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Separation (bp) Separation (bp) Separation (bp) Separation (bp)
Fig.S6
Contact frequency (P(s)) derived from Hi-C data obtained from CDK1as synchronized DT40
cells. Contact frequency P(s) plots for all 4 sets of WT Hi-C libraries. Plots for individual
chromosome arms are shown in gray; the deviation from the mean decay is shown in light gray.
Table S2 describes each sample in detail.
Fig.S7
A Contact frequency P(s) plots for all WT chromosome arms combined

102
WT-set#1 log WT-set#2 G2
101 G2 5min
contact probability
G2 15min
100 10min 30min
30min 60min
10-1
30min
10-2 60min
10-3
10-4
102
WT-set#3 0m WT-set#4 (GFP sorted) G2
101 2m
contact probability
15min
5m
100 30min
7.5m
60min
10m
10-1
10-2
10-3
10-4
103 104 105 106 107 108 103 104 105 106 107 108
separation (bp) separation (bp)
B Scaling derivative plots for all WT chromosome arms combined

101 WT-set#1 WT-set#2
contact probability derivative
log G2
G2 5min
100 G2 15min
10min 30min
30min 60min
10-1 30min
60min
10-2
10-3
101
WT-set#3 0min WT-set#4 (GFP sorted) G2
2min 15min
100 5min
30min
7.5min
60min
10min
10-1
10-2
10-3
103 104 105 106 107 108 103 104 105 106 107 108
Fig.S7
P(s) plots and their corresponding derivative plots. P(s) plots and their corresponding derivative
plots for 4 separate timecourse sets are shown for Hi-C datasets obtained from WT CDK1as cells
at indicated time points after release from G2 arrest.
Fig.S8 Chromosome 7 Hi-C interaction maps from all individual libraries for WT
0
log G2 WT
normalized reads
Set #1
7
70
0.10
EV1
0
-0.10
insulation
2
0
-2

0
normalized reads
Set #1
7
70
0.10
EV1
0
-0.10
insulation
2
0
-2

0
normalized reads
Set #2
7
70
0.10
EV1
0
-0.10
insulation
2
0
-2
WT-0min-S3-R1 WT-2.5min-S3-R1 WT-5min-S3-R2 WT-7.5min-S3-R1 WT-10min-S3-R2

0
normalized reads
Set #3
7
70
0.10
EV1
0
-0.10
insulation
2
0
-2
#21 WT-G2-S4-R4 #22 WT-2s15min-S4-R2 #23 WT-30min-S4-R4 #24 WT-60min-S4-R3

0
G2 15min 30min 60min
Set #4 (GFP sorted)
normalized reads
7
70
0.10
EV1
0
-0.10
2
nsulation
0
-2
Fig.S8
Hi-C data obtained from CDK1as synchronized DT40 cells at different time points in mitosis.
Hi-C interaction maps were generated for chromosome 7 normalized to 1,000,000 interactions.
Library names are indicated in the chromosome drawing on top right. Table S2 describes each
sample in detail. The top plot below each Hi-C interaction map displays compartment signal
(Eigenvector 1). The bottom graph shows insulation score (TADs). Data for 4 independent sets
of Hi-C data obtained from independent time courses are shown.
Fig.S9
Saddle plots for all individual chromosome arms from WT
WT
#1 EV1 rank #7 EV1 rank
1 .0 1 .0 EV1 rank
log G2 20% 20%
Set #1
0 .5 0 .5 strongest strongest
lo g 2 o b s / e x p
B-B B-A
ranked EV1 of WT-G2 (100kb)
1.76 0 .0 1.97 0 .0 median
= strength
30 bins with 3% genome
5th-95th percentile
median chr > 50Mb (!=chr1+chrZ)
− 0 .5 − 0 .5
EV1 rank
20% 20%
EV1 rank
strongest strongest
− 1 .0 − 1 .0
A-B A-A
− 0 .3
0 .6
0 .3
0 .0
G 2 e ig W T -9 0 G 2 -R 2 G 2 e ig
A A = 1 .5 6 , B B = 1 .2 1 , A A B B = 1 .3 7 , A B = 0 .6 6 , ( A A + B B ) /2 A B = 2 .0 8
0 .6
0 .3
0 .0
− 0 .3 EV1 rank EV1 rank EV1 rank EV1 rank EV1 rank
1 .0
1.0
Set #1
0 .5 0.5
log2 obs/exp
2.05 0 .0
1.16 1.03 1.05 1.02 0.0
− 0 .5 −0.5
EV1 rank
− 1 .0 −1.0
G 2 e ig

EV1 rank EV1 rank EV1 rank EV1 rank EV1 rank
1.0
0.5
Set #2
log2 obs/exp
2.78 1.44 1.19 1.13 1.08 0.0
−0.5
EV1 rank
−1.0
WT-0min-S3-R1 WT-2.5min-S3-R1 WT--5min-S3-R2 WT-7.5min-S3-R1 WT-10min-S3-R2

1.0
0.5
Set #3
log2 obs/exp
1.96 1.76 1.48 1.20 1.17 0.0
−0.5
EV1 rank
−1.0
WT-G2-S4-R4 WT-15min-S4-R2 WT-30min-S4-R4 WT-60min-S4-R2

EV1 rank EV1 rank EV1 rank EV1 rank
1.0
Set #4 (GFP sorted)
0.5
log2 obs/exp
1.59 1.06 1.07 1.08 0.0
−0.5
EV1 rank
−1.0
Fig.S9
Quantification of compartment interactions by “saddle plots”. The heatmaps show the average
distance-normalized cis interaction frequencies between pairs of 100kb bins as a function of their
compartment state (defined as the genome-wide rank of their eigenvector 1 value). The upper left
corner of the map shows the interactions between pairs of bins, where both belong to the inactive
compartment (i.e. compartment B); the lower right corner shows the interactions between pairs
of bins from the active compartment (compartment A). The number on each heatmap indicates
the overall strength of compartmentalization, defined as the median signal from the top 20% of
A-A and B-B interactions, divided by the top 20% of A-B interactions ((AA+BB)/2AB). Library
names are located on the top left. Table S2 describes each sample in detail.
Fig.S10
Normalized insulation Schematic of pileup at TAD borders
0.15 WT
0.1
log2 normalized score
0.05
0
-0.05
-0.1 G2 10min
-0.15 0min 15min
2.5min 30min
-0.2
5min 60min
-0.25
7.5min log
-0.3
+3
+4
-5
-4
-3
-2
-1
0b
+1
+2
00
00
00
00
00
00
00
00
00
p
kb
kb
kb
kb
kb
kb
kb
kb
kb
60
-500 Kb 0 +500 Kb
average
Sum of columns
of sums
Fig.S10
Average insulation profiles around WT G2 TAD boundaries on chromosome 7 at different time
points in mitosis The schematic on the right outlines how average insulation plots are calculated
around TAD boundaries. The insulation plot on the left shows the sum of the interactions for
each 50kb bins located 500kb upstream or downstream of a TAD border, normalized by the
average interactions for each timepoint. The presence of a minimum at 0 kb indicates a TAD
boundary. As cells progress through mitosis the minimum disappears indicating loss of
insulation and loss of the TAD boundary.
Fig.S11
Heatmaps from WT-20150609-R1 libraries showing 2nd Diagnal at similar length scales for chromosomes of a range of sizes

chromosome 2 (1.5x 108 bp)
chr2 chr2 chr2 chr2 chr2
0
chr2
149.6 Mb
70
Mb 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
chromosome 10 (2x 107 bp)

0
chr10
20.2 Mb 70
Mb 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20

#35 chr17 chr17 chr17 chr17 chr17
G2 5min 15min 30min 60min 0

chr17
11.0 Mb 100
Mb 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20

G2 5min 15min 30min 60min 0
chr21
6.9 Mb 150
Mb 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
101 WT-G2-S2-R3 WT-5min-S2-R1 WT-15min-S2-R1 WT-30min-S2-R3 WT-60min-S2-R2 chr2

100 chr10
chr17
Contact probability
10-1 chr21
10-2
10-3
10-4
10-5
10-6
104 105 106 107 108 104 105 106 107 108 104 105 106 107 108 104 105 106 107 108 104 105 106 107 108
separation (bp) separation (bp) separation (bp) separation (bp) separation (bp)
Fig.S11
Position of the second diagonal is independent of chromosome size. . We compared contact
frequency plots (P(s)) for wild-type chromosomes 2 (150 Mb), 10 (20 Mb), 17 (11 Mb) and 21
(6.8 Mb) to illustrate that the position of the second diagonal does not depend on chromosome
size. At the onset of mitosis (G2) and in prophase (t = 5 min), there is no second diagonal for any
chromosome. In early prometaphase (t = 15min), a second diagonal (increase in contact
frequency) can be seen for all chromosome sizes and in each Hi-C interaction map. A t = 15
minutes the position of the second diagonal is smaller than the size of each of these chromosome.
At t = 30 minutes, the position of the second diagonal moved to larger genomic distance (~ 7
Mb). This distance exceeds the size of chromosome 21 (> 6.8 Mb), and leads to a disappearance
of a visible increase in contact frequency for this chromosome only (as is observed in in both the
Hi-C interaction map and contact frequency plot below). At t = 60 minutes the position of the
second diagonal (~12 Mb) exceeds the size of chromosome 17, so that the second diagonal is no
longer detected along that chromosomes. P(s) plots (bottom) illustrate that positions of the
second diagonals are the same for all chromosomes, at different timepoints in prometaphase.
Fig.S12
A Heatmaps, compartments and TADs for HelaS3

HelaS3 CCL2.2 HelaS3 CCL2.2
0 0
log PM 98%
normalized reads
normalized reads
14 10 14 100
0.10 0.10
insulation EV1
0 0
-0.10 -0.10
2 2
0 0
-2 -2
B Contact frequency P(s) plot for HelaS3

101
log
PM98
contact probability
100
P(s) ~ s-0.5
10−1
10−2
10kb 100kb 1Mb 10Mb 100Mb
separation
Fig.S12
Hi-C interaction maps from HeLaS3 cells arrested in late prometaphase display a second
diagonal. (A) Hi-C interaction maps for HelaS3 (ATCC CCL-2.2). Hi-C was performed on
logarithmically growing HelaS3 cells (log) and HelaS3 cells arrested in late prometaphase with
nocodazole, resulting in 98% prometaphase (PM 98%). Nocodazole synchronization was
performed for 3 hrs following a double thymidine block, as described before (8). The
compartment and insulation signals below the interaction frequency heatmaps are as in Fig. S8.
(B) P(s) for HelaS3 logarithmically growing cells (log) and for prometaphase arrested cells
(PM98) for chromosome 14, as in Fig. 2. The dotted line indicates P(s) ~ s-0.5 reported before for
mitotic chromosomes (8). The local peak in P(s) around 10 Mb corresponds to the second
diagonal band visible in the Hi-C interaction map (A).
Fig.S13
Average Hi-C maps around condensin binding sites and loop anchor in models
WT-60 min SMC2 peaks (p<103) WT-60 min CAP-H peaks (p<103) simulated average at loop anchor
-1000 1.0
log2 obs/exp
0 0
1000 -1.0
-1000 0 1000 -1000 0 1000 -1000 0 1000
relative position (kb) relative position (kb) relative position (kb)
Fig.S13
The Hi-C contact footprint of condensin binding in experimental and simulated Hi-C maps. (A-
B) The Hi-C maps of 60-min mitotic chromosomes averaged over 2Mb regions around (A)
13120 SMC2 and (B) 4674 CAPH ChIP-seq peaks. (C) The simulated Hi-C map of the 60-min
mitotic chromosome model averaged over 2Mb regions around 1000 loop anchors. All three
maps were normalized for the distance-dependent contact frequency decay in the corresponding
dataset. See Supplementary Materials for description and interpretation of this analysis.
Fig.S14
A Second diagonal not explained by sister overlap

101
inter-axial distance / chromatid R:
contact probability
100
0.00
0.25 0 nm/
0.50 0 Mb
0.75 z-axis
10-1 1.00
1.25
1.50
1.75
2.00
WT-30min
10-2
104 105 106 107
separation (bp)
B Without nested loops it does not fit

WT-15min WT-30min
101
loops 100/0kb, loops 200/0kb,
spiral: period 5Mb, radius 154nm, step 250nm spiral: period 20Mb, radius 315nm, step 250nm
contact probability
100
P(s) ~ s-0.5
P(s) ~ s0.5
10-1 WT-60min
loops 200/0kb,
spiral: period 20Mb, radius 315nm, step 250nm
loops 200/0kb,
loops 100/0kb,
10-2
104 105 106 107 104 105 106 107 104 105 106 107
separation (bp) separation (bp) separation (bp)
C An external helix does not fit
101 WT-30min
120/40kb, 8Mb, step 150nm, R_helix=210nm, R_chromatid=337nm
contact probability
100
P(s) ~ s-0.5
10-1
10-2
104 105 106 107
separation
Fig.S14
Rejected polymer models of prometaphase chromosomes. (A) Interactions between sister
chromatids do not produce a second diagonal in Hi-C interaction maps. Plot shows P(s) of a
model of sister chromatid interactions, where two identical prophase models are overlaid on top
of each other in 3D space. The P(s) in this model was calculated under the assumption that Hi-C
would not be able distinguish between the two copies of the same locus on the two sister
chromatids. P(s) plots were calculated for a different levels of sister chromatid overlap. (B)
Without nested loops polymer models do not reproduce experimental Hi-C data. Plots show P(s)
of prometaphase models without nested loops. Left-right: the panels show the best fitting models
for each time point (t = 15,30 and 60 minutes) in prometaphase. (C) Simulation of an external
helix does not produce P(s) that corresponds to experimental prometaphase Hi-C data. The P(s)
curves of examples of prometaphase models with wide spiraling of the scaffold (i.e. the radius of
the scaffold > ½ of the chromatid radius). All panels: black lines - experimental Hi-C data
(dataset indicated in the legend); colored lines - P(s) plot obtained by simulations with different
parameters (indicated in the legend).
Fig.S15
Gyre visualization and quantification for chromosome 2
A SMC2-mAID CAP-H-mAID CAP-H2-mAID WT

Note:
No auxin, asynchronous cells
100ng/ml cocemid for 2.5h
75 mM KCl for 5 min
Me/Ac fixation
drop/dry on slides
Vectashield with DAPI
1 µm
B chromosome 2 chromosome 2 chromosome 2 chromosome 2

10 gyres 10 gyres? 9 gyres 6 gyres?
149.6 Mb 149.6 Mb 149.6 Mb 149.6 Mb
=15 Mb/gyre =15 Mb/gyre = 16.6 Mb/gyre = 24.9 Mb/gyre
length: 5.6 µm length: 6.0 µm length: 5.2 µm length: 4.5 µm
C SMC2-mAID CAP-H-mAID
2 µm
CAP-H2-mAID WT
Fig.S15
Gyre visualization and quantification for chromosome 2. (A) Visualization (by DAPI) of a
repeating structure on DT40 mitotic chromosomes following hypotonic treatment and spreading
of indicated DT40 cell lines treated with colcemid for 2.5 hours. (B) Gyre count and size per
gyre (Mb) estimates for chromosome 2. (C) Chromosome spreads of indicated cell lines. Boxed
chromosomes are chromosome 2 based on size and centromere position, used for measurements
in (A) and (B).
Fig.S16
GFP expression of condensins
A Anaphase
CAP-H overlay GFP DNA
1 µm
1 µm
B Prometaphase
CAP-H-mAID CAP-H2-mAID SMC2-mAID

overlay GFP DNA overlay GFP DNA overlay GFP DNA
Side view
(rare)
1 µm
Top view CAP-H

(majority) overlay GFP DNA
Fig.S16
Helical distribution of SMC2-mAID-GFP, CAP-H-mAID-GFP and CAP-H2-mAID-GFP in
single sister chromatids. (A) Distribution of condensin components (as visualized by GFP) in
anaphase chromosomes where only a single sister chromatid is present (this simplifies
interpretation of the image). (B) Distribution of condensin components in rare views of
prometaphase chromatids in which the two sister chromatids were oriented normal to the surface
of the slide. This enabled us to use optical sectioning to resolve a single sister chromatid. Scale
bars 1 µm. DNA is visualized by DAPI staining.
Fig.S17
Chromosome length and width measurements
9
30min
8
60min MG132
7
60min NOC
6
5
4
3
2
1
0
Length (μm) width (μm)
Fig.S17
Chromosome length and width measurements. Measurements of the length and width of
chromosome 1 (196 Mb) in DT40 cells prepared under the conditions used for Hi-C experiments,
at different timepoints in prometaphase.
Fig.S18
P r o p hase and p ro m e t a p h as e fi ts
f or l o op leng t h a n d d e n sit y
Loop length (kb)
6 00
4 00
2 00
60
Linear chromatin
dens it (M b/µ m )
40
20
2 5 7 10 15 30 60
Ti m e ( m i n )
Fig.S18
Parameters of the best fitting models as a function time after release from G2 arrest. Chromatin
density and loop length increase linearly from prophase through prometaphase.
Fig.S19
Western blots for auxin-treated condensin mutants
no auxin auxin
G2 M G2 M
SMC2-mAID
α- Tubulin
CAP-H2-mAID
α- Tubulin
CAP-H-mAID
α- Tubulin
Fig.S19
Western blots showing protein levels of SMC2-mAID, CAP-H-mAID and CAP-H2-mAID for
corresponding cell lines treated with or without auxin induction. Western blots indicate SMC2-
mAID, CAP-H2-mAID and CAP-H-mAID are efficiently depleted after 3 hours of auxin
addition.
Fig.S20 A Chromosome 7 Hi-C interaction maps from all individual libraries for SMC2-mAID plus auxin
SMC2-mAID-G2-R1 SMC2-mAID-10min-S1-R1 SMC2-mAID-45min-S1-R1 SMC2-mAID-75min-R1
0 0
G2 SMC2-mAID 10min 45min 75min
normalized reads
normalized reads
Set #1 (GFP sorted)
7
70 70
0.10 0.10
EV1
EV1
0 0
-0.10 -0.10
insulation
2 2
insulation
0 0
-2 -2
SMC2-mAID-G2-R2 SMC2-mAID-G2-S2-R3 SMC2-mAID-10min-S2-R2 SMC2-mAID-45min-S2-R2 SMC2-mAID-75min-S2-R2

0
G2 G2 10min 45min 75min
normalized reads
Set #2
7 70
0.10
EV1
0
-0.10
2
insulation
0
-2
B Chromosome 7 Hi-C interaction maps from all individual libraries for CAP-H2mAID plus auxin
CAP-H2-mAID-G2-S1-R1 CAP-H2-mAID-G2-S1-R1
0
G2 G2 CAP-H2-mAID
normalized reads
Set #1
7
20
0.10
EV1
0
-0.10
insulation
2
0
-2
CAP-H2-mAID-8.5min-S1-R1 CAP-H2-mAID-10min-S1-R1 CAP-H2-mAID-15min-S1-R1 CAP-H2-mAID-30min-S1-R1 CAP-H2-mAID-60min-S1-R1

0
8.5min 10min 15min 30min 60min
normalized reads
Set #1
7
20
0.10
EV1
0
-0.10
insulation
2
0
-2
0
G2 G2
normalized reads
Set #2
7
70
0.10
EV1
0
-0.10
2
insulation
0
-2
CAP-H2-mAID-7.5min-S2-R2 CAP-H2-mAID-10min-S2-R2 CAP-H2-2mAID-15min-S2-R2 CAP-H2-mAID-30min-S2-R2 CAP-H2-mAID-60min-S2-R2

0
normalized reads
Set #2
7 70
0.10
EV1
0
-0.10
2
insulation
0
-2 -2
C Chromosome 7 Hi-C interaction maps from all individual libraries for CAP-H-mAID plus auxin
CAP-H-mAID-G2-S1-R1 CAP-H-mAID-G2-S1-R1
0
G2 G2 CAP-H-mAID
normalized reads
Set #1
7
70
0
2
0
-2
CAP-H-mAID-8.5min-S1-R1 CAP-H-mAID-10min-S1-R1 CAP-H-mAID-15min-R1 CAP-H-mAID-30min-S1-R1 CAP-H-mAID-60min-S1-R1

0
normalized reads
Set #1
7
70
0.10
EV1
0
-0.10
2
insulation
0
-2
0
G2 G2
normalized reads
Set #2
7
70
0.10
EV1
0
-0.10
insulation
2
0
-2
CAP-H-mAID-7.5min-S2-R2 CAP-H-mAID-10min-S2-R2 CAP-H-mAID-15min-S2-R2 CAP-H-mAID-30min-S2-R2 CAP-H-mAID-60min-S2-R2

0
normalized reads
Set #2
7 70
0.10
EV1
0
-0.10
2
insulation
0
Fig.S20
Hi-C interaction maps obtained from SMC2, CAP-H and CAP-H2 depleted cells at different
timepoints in mitosis. Hi-C interaction maps were generated for chromosome 7, normalized to
1,000,000 interactions. Dataset names are indicated in the chromosome drawing on top. Table
S2 describes each sample in detail. (A): SMC2-mAID cells; (B): CAP-H2-mAID cells; (C):
CAP-H-mAID cells. The top plot below each Hi-C interaction map displays compartment signal
(Eigenvector 1; with percentage signal explained by first eigenvector). The bottom graph shows
insulation score (TADs). Data for two independent time courses are shown.
Fig.S21
DAPI stain for synchronized cells
G2 Prophase NEBD Prometaphase
G2 2.5 min 5 min 7.5 min 10 min 15 min 30 min 60 min (MG) 60 min (Noc)
wt
5 µm
SMC2-mAID
CAPH2-mAID
CAPH-mAID
Fig.S21
Microscopy analysis of chromosome morphology for wild-type (WT) cells and SMC2, CAP-H
and CAP-H depleted cells. Chromosomes were stained with DAPI. Samples are ordered by time
of harvest after release from G2 arrest. NEBD was used a marker for the prophase-prometaphase
transition. Cells were prevented from entering anaphase by addition of nocodazole or MG132.
Scale bar indicates 5 micron. Noc: nocodazole; MG: MG132.
Fig.S22
A Saddle plots for all individual chromosome arms from SMC2-AID plus auxin
SMC2-mAID
SMC2-G2-S1-R1 SMC2-10min-S1-R1 SMC2-45min-S1-R1 SMC2-75min-S1-R1
EV1 rank EV1 rank EV1 rank EV1 rank
1.0 1.0
Set #1 (GFP sorted)
0.5 0.5
log2 obs/exp
log2 obs/exp
2.33 0.0 1.73 1.24 1.20 0.0
−0.5 −0.5
EV1 rank
−1.0 −1.0
SMC2-G2-S2R2 SMC2-G2-S2-R3 SMC2-10min-S2-R2 SMC2-45min-S2-R2 SMC2-75min-S2-R2

1.0
0.5
Set #2
log2 obs/exp
1.72 1.95 1.57 1.15 1.16 0.0
−0.5
EV1 rank
−1.0
B Saddle plots for all individual chromosome arms from CAP-H2-mAID plus auxin
CAP-H2-mAID
CAP-H2-G2-S1-R1 CAP-H2-G2-S1-R1
EV1 rank EV1 rank
1.0
G2 G2
Set #1
0.5
log2 obs/exp
2.05 2.05 0.0
−0.5
EV1 rank
−1.0
CAP-H2-8.5min-S1-R1 CAP-H2-10min-S1-R1 CAP-H2-15min-S1-R1 CAP-H2-30min-S1-R1 CAP-H2-60min-S1-R1

1.0
0.5
Set #1
log2 obs/exp
1.52 1.45 1.19 1.09 1.08 0.0
−0.5
EV1 rank
−1.0
CAP-H2-G2-S2-R2 CAP-H2--G2-S2-R1
EV1 rank EV1 rank
1.0
G2 G2
0.5
Set #2
log2 obs/exp
2.06 2.36 0.0
−0.5
EV1 rank
−1.0
CAP-H2-7.5min-S2-R2 CAP-H2-10min-S2-R2 CAP-H2-15min-S2-R2 CAP-H2-30min-S2-R2 CAP-H2-60min-S2-R2

1.0
0.5
Set #2
log2 obs/exp
1.37 1.29 1.12 1.06 1.05 0.0
−0.5
EV1 rank
−1.0
C Saddle plots for all individual chromosome arms from CAP-H-mAID plus auxin
CAP-H-mAID
CAP-H-G2-S1-R1 CAP-H-G2-S1-R1
EV1 rank EV1 rank
1.0
G2 G2
Set #1
0.5
log2 obs/exp
2.32 2.32 0.0
−0.5
EV1 rank
−1.0
CAP-H-8.5min-S1-R1 CAP-H-10min-S1-R1 CAP-H-15min-S1-R1 CAP-H-30min-S1-R1 CAP-H-60min-S1-R1

1.0
0.5
Set #1
log2 obs/exp
1.23 1.19 1.14 1.08 1.09 0.0
−0.5
EV1 rank
−1.0
CAP-H-G2-S2-R2 CAP-HG2n-S2-R2
EV1 rank EV1 rank
1.0
G2 G2
0.5
Set #2
log2 obs/exp
2.37 2.51 0.0
−0.5
EV1 rank
−1.0
CAP-H-7.5min-S2-R2 CAP-H-10min-S2-R2 CAP-H-15min-S2-R2 CAP-H-30min-S2-R2 CAP-H-60min-S2-R2

1.0
Set #2
0.5
log2 obs/exp
1.24 1.12 1.09 1.07 1.06 0.0
−0.5
EV1 rank
−1.0
Fig.S22
Quantification of compartment interactions in SMC2, CAP-H and CAP-H2 depleted cells by
saddle plots. (A): SMC2-mAID cells; (B): CAP-H2-mAID cells; (C): CAP-H-mAID cells. See
supplementary Materials and legend of Fig. S9 for description of saddle plots.
Fig.S23
Normalized insulation at TAD boundaries, chromosome 7 in condensin mutants plus auxin
0.15 SMC2-mAID CAP-H2-mAID CAP-H-mAID

log2 normalized score
0.1
0.05
0
-0.05
G2 G2
7.5min 7.5min
-0.1
G2 10min 10min
-0.15
10min 15min 15min
-0.2
45min 30min 30min
-0.25
75min 60min 60min
-0.3
-5
-4
-3
-2
-1
0b
+1
+2 b
+3 b
+4
+5
-5
-4
-3
-2
-1 b
0b
+1
+2 b
+3 b
+4
+5
-5
-4
-3
-2
-1 b
0b
+1
+2 b
+3 b
+4
+5
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
p
p
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
k
kb
kb
kb
kb
kb
kb
kb
Fig.S23
Average insulation profiles around G2 TAD boundaries on chromosome 7 for cells depleted for
SMC2, CAP-H2 or CAP-H at different timepoints in mitosis. Plots show the sum of the
interactions for each 100 kb bin 500kb upstream or downstream of the TAD normalized by the
average sum of interactions for each time point. The presence of a minimum at 0 kb indicates a
TAD boundary. SMC2 depletion prevents complete loss of the minimum, indicated maintenance
of TAD boundaries. Depletion of CAP-H2 leads to slower loss of TAD boundaries as compared
to WT (Fig. S10). Depletion of CAP-H does not prevent efficient loss of TAD boundaries.
Fig.S24 A Contact frequency P(s) plots for all individual chromosome arms plus auxin
SMC2-mAID-G2-S1-R1 SMC2-mAID-10min-S1-R1 SMC2-mAID-45min-S1-R1 SMC2-mAID-45min-S1-R1

2 2
10 10
G2 SMC2-mAID 10min 45min 75min
Set #1 (GFP sorted)
contact frequency
contact frequency
1 1
10 10
0 0
10 10
-1 -1
10 10
-2 -2
10 10
-3 -3
10 10
-4 -4
10 10
3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Separation (bp) Separation (bp) Separation (bp) Separation (bp)
SMC2-mAID-G2-S2-R2 SMC2-mAID-G2-S2-R3 SMC2-mAID-10min-S2-R2 SMC2-mAID-45min-S2-R2 SMC2-mAID-75min-S2-R2

2
10
contact frequency
1
10
0
10
Set #2
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
B CAP-H2-mAID contact frequency P(s) plots for all individual chromosome arms plus auxin
2
10
G2 G2 CAP-H2-mAID
contact frequency
1
10
0
Set #1
10
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10
CAP-H2-mAID-8.5min-S1-R1 CAP-H2-mAID-10min-S1-R1 CAP-H2-mAID-15min-S1-R1 CAP-H2-mAID-30min-S1-R1 CAP-H2-mAID-60min-R1

2
10
contact frequency
1
10
0
10
Set #1
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
2
10
G2 G2
contact frequency
1
10
0
10
Set #2
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10
CAP-H2-mAID-7.5min-S2-R2 CAP-H2-mAID-10min-S2-R2 CAP-H2-mAID-15min-S2-R2 CAP-H2-mAID-30min-S2-R2 CAP-H2-mAID-60min-S2-R2

2
10 7.5min 10min 15min 30min 60min
contact frequency
1
10
0
10
Set #2
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
C CAP-H contact frequency P(s) plots for all individual chromosome arms plus auxin
2
10
G2 G2 CAP-H-mAID
contact frequency
1
10
0
Set #1
10
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10

2
contact frequency
1
10
0
10
Set #1
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
2
10 G2 G2
contact frequency
1
10
0
10
Set #2
-1
10
-2
10
-3
10
-4
10
10
3
10
4
10
5
10
6
10
7
10
8
10
3
10
4
10
5
10
6 10
7
10
8

2
contact frequency
1
10
0
10
Set #2
-1
10
-2
10
-3
10
-4
10 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Fig.S24
Contact frequency (P(s)) derived from Hi-C data obtained from CDK1as synchronized DT40
cells depleted for SMC2, CAP-H2 or CAP-H. (A): SMC2-mAID cells; (B): CAP-H2-mAID
cells; (C): CAP-H-mAID cells. Contact frequency P(s) plots are shown for all Hi-C datasets (two
independent time courses). Plots for individual chromosome arms are shown in gray; the
deviation from the mean decay is shown in magenta (SMC2), red (CAP-H2-mAID) or blue
(CAP-H-AID).
Fig.S25
Contact frequency P(s) plots for all chromosome arms combined from condensin mutants plus auxin
102
SMC2mAID-set#1 (GFP sorted) G2 SMC2-set#2 G2
101 10m 10m
contact probability
45m 45m
100
75m 75m
10-1
10-2
10-3
10-4
102
CAP-H2mAID--set#1 G2 CAP-H2-mAID-set#2 G2
101 8.5m 7.5m
contact probability
10m 10m
100 15m 15m
30m 30m
10-1
60m 60m
10-2
10-3
10-4
102
CAP-HmAID-set#1 G2 CAP-H-mAID-set#2 G2
contact probability
101 8.5m 7.5m

10m 10m
100
15m 15m
10-1 30m 30m
60m 60m
10-2
10-3
10-4
103 104 105 106 107 108 103 104 105 106 107 108
Fig.S25
P(s) plots for SMC2-mAID, CAP-H2-mAID and CAP-H-mAID DT40 cells at different time
after degron induction and release from G2 arrest. P(s) plots for Hi-C datasets corresponding to
different timepoints in each time course experiment are plotted in one graph.
Fig.S26
Contact frequency P(s) derivative plots for all chromosome arms combined from condensin mutants plus auxin
101
SMC2-mAID-set#1 (GFP sorted) SMC2-mAID-set#2
G2 G2
10min 10min
100 45min 45min
75min 75min
10-1
10-2
10-3
101
CAP-H2-mAID-set#1 G2 CAP-H2-mAID-set#2 G2
8.5min 7.5min
100 10min 10min
15mn 15min
30min 30min
10-1
60min 60min
10-2
10-3
101
CAP-H-mAID-set#1 CAP-H-mAID-set#2
G2 G2
8.5min 7.5min
100
10min 10min
15min 15min
10-1 30min 30min
60min 60min
10-2
10-3
103 104 105 106 107 108 103 104 105 106 107 108
Figure S26
Derivatives of P(s) plots for SMC2, CAP-H2 or CAP-H depleted cells. Plots show derivative of
P(s) plots for Hi-C datasets obtained from SMC2-mAID, CAP-H2-mAID and CAP-H-mAID
CDK1as cells at indicated time points after auxin-induced protein depletion and release from G2
arrest.
Fig.S27
interphase 5 min prophase 7.5 min prophase
coverage = 30% coverage = 88% coverage = 96%

= 169 kb = 46 kb = 52 kb
CTCF
A
TADs
COMPs
0
log(contact frequency, a.u.)

Hi-C
B -2.8
1
0
EV1
-1
100 kb, var=0.160 100 kb, var=0.039 100 kb, var=0.037
1 250 kb, var=0.096 250 kb, var=0.007 250 kb, var=0.005
insulation
-1
EV1 rank EV1 rank EV1 rank
C 1
compartmentalization
log2(obs/exp)
2.7 1.6 1.5
EV1 rank
EV1 rank
EV1 rank
-1
D 500 0
log10(cont. freq., a.u.)

kb
boundary pileup
0 -2
500 kb 0 500 kb 500 kb 0 500 kb 500 kb 0 500 kb
E
example conformation
Figure S27
Chromosome compaction by loop extrusion suppresses TADs and compartments even in the
presence of CTCF. A possible mechanism for the loss of TADs (gray rectangles in (A) and
insulation signal in (B)) and A/B compartments in prophase is revealed by polymer simulations
with loop extrusion and compartmental interaction. In interphase, the chromatin fiber is locally
compacted into TADs by sparse loop extrusion factors (LEFs, yellow). Compartmentalization
(yellow/magenta COMPs in (A) and EV1 in (B, C)) emerges from preferential interactions
between loci of the same compartment type. In prophase, coverage of chromosomes by LEFs
(red) increases strongly. According to this model, TADs disappear in prophase because CTCF
sites are now just one of many loci where loops are stacked up against each other and contacts
across them are as frequent as between them (TAD boundary pileup in (D)),
compartmentalization is suppressed (compartmentalization plots in (C)), because the bottlebrush
imposes linear ordering and reduces fiber flexibility. Example conformation renderings are
shown in (E).
Tables S1-S4
Table S1
Sample and FACS summary. All samples with their cell type used for Hi-C libraries
(name_galGal5) are listed by the names used throughout all figures. For each cell collection
(time), the main cell cycle phase, treatments and drugs used are listed. FACS analysis was used
to determine AID-GFP presence (and level of protein degradation). A subset of cells was sorted
(sorted) for GFP positivity or negativity respectively, as indicated.
Table S2
Hi-C dataset overview. The names used corresponds to those used in figures and Table S1. Each
harvested cell sample was submitted to Hi-C as part of a set (Set#) and the sequencing results
and mapping statistics for galGal5 (gg5) are listed. Valid pair: reads mapped for both ends;
NRVP (non-redundant valid pairs): valid pairs with duplicate reads removed (see Supplementary
Methods); %Dangling: percentage of dangling ends (inward reads [|] derived from false
biotin incorporation); %cis: percentage of intra-chromosomal interactions; %redundant:
percentage of exact (PCR) duplicate reads (used to determine NRVP).
Table S3
DNA constructs used for generation of cell lines. Mutant: name used throughout the manuscript;
background: cell line used to generate modified cell line; construct: vector used to generate
mutants; FBOX: the specific FBOX protein used to degrade the AID-fused mutant; endogenous:
the modification to the endogenous locus using homologous recombination (HR) or CRISPR;
alleles: the number and location of altered alleles in DT40 mutants; selection: antibiotic
resistance used for mutant selection; publication: previous work using these mutants.
Table S4
Short- and long-distance parts of the P(s).Equalization of the contribution of the short- and long-
distance parts of the P(s) was calculated using the r.m.s. P(s) difference in each of these regions
separately and reporting the mean of the two.
Movie S1
The emergence of nested loop architecture in loop extrusion lattice simulations of slow- and fast-
exchanging condensin populations. The video shows the simulated dynamics of loops extruded
by condensins I and II on a 2Mb chromatin segment during two hours of simulated time. The
positions of loops formed by fast-exchanging condensins I are shown as blue semi-transparent
arcs and those formed by slow-exchanging condensin II are shown in red. The detailed
description of simulations is available in Supplemental Materials and Methods.
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Supplementary Materials for

Published 18 January 2018 on Science First Release

This PDF file includes:

Materials and Methods

Other Supplementary Material for this manuscript includes the following:

Time course experiments

Modifications to the time course experiment

Cell sorting to isolate cells depleted of target proteins

Nocodazole addition to prevent anaphase entry

Microscopy on fixed cells

Fixation of chromosomes from mitotic cells

Lamin B1 and Histone H3 Phos-Ser10 staining

Chromosome length measurements

Visualization of a repeating structure on chicken mitotic chromosomes

Helical distribution of GFP signal in single sister chromatids

Flow cytometry analysis

Chromatin Enrichment for Proteomics (ChEP)

ChIP-seq data re-analysis.

Hi-C data analysis

Mapping sequenced Hi-C reads

Hi-C based karyotyping

Contact probability (P(s)) curves

Binning and balancing of Hi-C data

Insulation analysis (TADs)

Contacts within loops

Contacts between loops

Randomly oriented uncorrelated loops

Loops with correlated orientation

We can derive the frequency of angular overlaps explicitly:

Loops with spiral staircase orientation

Polymer models of mitotic chromosomes

Simulations of prometaphase and CAP-H-depleted chromosomes

Simulated Hi-C and P(s) calculation for polymer models

Model selection via comparison of simulated and experimental P(s) curves

P(s) estimation for prophase models with sister chromatid cohesion

Estimation of the rate of loop formation at condensin binding sites.

Simulations of nested loop formation by loop extrusion.

To simulate extrusion of loops by condensins I and II, we considered 20Mb of chromatin

Simulations of suppression of TADs and compartments by loop extrusion in prophase

A DT40 karyotype Chromosomes galgal5

B Integration sites C Translocations

80 63 SOX5 68.5 107 MIR221 113 137 MYC 143

FACS analyis after release from synchronization

1.0K 1.0K 250

800 800 200

600 600 150

400 400 100

7696 8756 8854

5779 5854 5458 5712

0 200 400 0 200 400 0 200 400 0 200 400

G2 7.5 min 10 min

Interphase Prophase Prometaphase

A Images corresponding to samples for condensin ChEP data

G2 0 min 2.5 min 7.5 min 12 min

17 min 22 min 27 min 42 min 57 min 72 min

0.02 NEBD NEBD NEBD SMC2-mAID WT

A Insulation analysis at varying length scales B Insulation score variance